Protein disulfide isomerase and method of using

ABSTRACT

Protein disulfide isomerase, which catalyzes the exchange reaction between sulfhydryl and disulfide in a protein for formation of the most stable natural type disulfide bonds, is useful for formation of natural type disulfide bonds in a protein which is produced in a prokaryotic cell.

This is a continuation of application Ser. No. 07/982,138, filed on Nov.25, 1992, now U.S. Pat. No. 5,700,678, which is a continuation of U.S.Ser. No. 07/635,812, filed Jan. 2, 1991, now abandoned, which is acontinuation of U.S. Ser. No. 07/199,307, filed May 26, 1988, nowabandoned.

FIELD OF INDUSTRIAL APPLICATION

The present invention relates to a polypeptide and a method forproducing the polypeptide, and specifically relates to a polypeptidepossessing protein disulfide isomerase activity and a method forproducing the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematized cleavage maps of the cDNA with restrictionenzymes as cloned in pT3BP-1 and pT3BP-2 obtained in Reference Example2. In FIG. 1, (1) represents the cDNA as cloned in pT3BP-1 and (2)represents the cDNA as cloned in pT3BP-2. The abbreviations A, E, H, Pand S represent Aat II, EcoR I, Hinc II, Pvu II and Ssp I, respectively;▪ represents the portion corresponding to the amino acid sequencederived from tryptic peptide mapping for purified T3BP protein.

FIGS. 2A, 2B, 2C and 2D shows the base sequence of the cDNA as cloned inthe pT3BP-2 obtained in Reference Example 2 and the amino acid sequencededuced from the base sequence.

FIG. 3 shows the amino acid sequence of human PDI.

FIG. 4 shows a schematized cleavage map of the cDNA with restrictionenzymes as cloned in pT3BP-3 obtained in Example 1. In FIG. 4, theabbreviations B, C, E, H and P represent BamH I, Cla I, EcoR I, Hind IIIand Pst I, respectively.

FIGS. 5A and 5B shows the base sequence of the cDNA as cloned in pT3BP-3obtained in Example 1 and the amino acid sequence deduced from the basesequence.

FIG. 6 shows the construction scheme for the plasmid for expression ofthe human PDI gene in animal cells.

FIGS. 7A and 7B shows the results (fluorescent photomicrographs) ofdetection by the fluorescent antibody method of human PDI as synthesizedin COS-7 cells into which the human PDI gene were introduced. In FIG.7A, represents a fluorescent photomicrograph showing the morphology ofthe PDI gene-introduced cell and FIG. 7(B) represents a fluorescentphotomicrograph of the morphology of the cell without the geneintroduction as the control.

FIG. 8 shows the construction scheme for the plasmid for expression ofthe human PDI gene in Escherichia coli.

FIG. 9 shows the elution pattern of human PDI produced in Escherichiacoli as obtained by the immunoprecipitation method. In FIG. 9, the lanesB and D correspond to DH1/pTB766 and MM294/pTB766, respectively; thelanes A and C correspond to DH1/ptrp781 and MM294/ptrp781, respectively.

FIG. 10 shows the construction scheme for the plasmid for expression ofthe human PDI gene in yeasts.

FIG. 11 shows SDS-polyacrylamide gel electrophoresis of PDI. Lane 1 andLane 2 correspond to purified PDI and marker proteins, respectively.

FIG. 12 shows ultraviolet absorption spectrum of purified PDI.

PRIOR ART AND PROBLEMS TO BE SOLVED

It is generally known that formation of disulfide bonds is extremelyimportant in the retention of protein's configuration and the expressionof its activity. In eukaryotic cells, disulfide bonds are formed inendoplasmic reticula simultaneously with or immediately after proteintranslation J. Biol. Chem., 257, 8847 (1982)!. Such disulfide bonds canalso be non-enzymatically formed in vitro; however, the reaction rate islower than that in vivo, but also the conditions in vitro do not reflectthose in vivo. Based on this fact, Anfinsen et al. conjectured that aprotein named as disulfide interchange protein would function in theformation of disulfide bonds in vivo J. Biol. Chem., 238, 628 (1963)!.Although this conjecture still remains a hypothesis, enzymes which acton reduced and scrambled ribonucleases (both are inactive types) topromote the formation of true disulfide bonds for active ribonucleases,were discovered in various eukaryotes and purified Biochemistry, 20,6594 (1981); Biochem. J., 213, 225 (1983); Biochem. J., 213, 245(1983)!. The scrambled ribonuclease, which is prepared by reduction andre-oxidization under denaturing conditions, is a mixture of variousmolecular species formed as a result of random recombination of the 4pairs of disulfide bonds in the active ribonuclease Biochem. J., 213,235 (1983)!. The enzyme which converts an inactive ribonuclease to anactive ribonuclease, is named protein disulfide isomerase (PDI, EC5.3.4.1), and is a dimer comprising 2 identical subunits of a molecularweight of 52,000 to 62,000, its isoelectric point being 4.0 to 4.5Trends in Biochem. Sci., 9, 438 (1984); Methods in Enzymol., 107, 281(1984)!. The already-known protein disulfide reductase (also calledglutathione insulin transhydrogenase, EC 1.8.4.2.) is thought of asidentical with PDI Eur. J. Biochem., 32, 27 (1973); Biochemistry, 14,2115 (1975)!.

In 1985 Edman et al. succeeded in cloning a complementary DNA of ratliver PDI and determined its entire base sequence Nature, 317, 267(1985)!. Rat liver PDI comprises 489 amino acids with a signal peptideconsisting of 19 amino acids being added to its amino terminal. It alsohas regions homologous with Escherichia coli thioredoxin Eur. J.Biochem., 6, 475 (1968); Proc. Natl. Acad. Sci. USA, 72, 2305 (1975);Methods in Enzymol., 107, 295 (1984)!, which is known as a cofactor forvarious oxidation-reduction reactions such as ribonucleotide reductasereaction, in two sites, namely in the vicinities of the amino terminaland carboxyl terminal. Each region includes a homologous sequence having2 Cys (Trp-Cys-Gly-Cys-Lys), which is considered to catalyze PDIreaction via exchange between disulfide and sulfhydryl. It has alsorecently been reported that Escherichia coli thioredoxin exhibits PDIactivities in the presence of reduced or scrambled ribonuclease as thesubstrate Proc. Natl. Acad. Sci. USA, 83, 7643 (1986)!.

The utility of PDI may include its application to refolding ofrecombinant proteins produced by Escherichia coli. Due to recent rapidprogress in genetic engineering technology, mass production ofeukaryotic proteins using Escherichia coli has become possible; however,the recombinant proteins thus obtained may lack true disulfide bonds, ormay have misformed disulfide bonds EMBO Journal, 4, 775 (1985); inGenetic Engineering, Vol. 4 (edited by Williamson, R.), pp. 127 (1983)!.A refolding procedure is therefore required to obtain an activerecombinant protein having true disulfide bonds. This procedure has beenchemically performed by oxidation-reduction reaction using a redoxbuffer, etc.; however, it appears that the use of PDI, which mayfunction in vivo, is more advantageous in specific formation of truedisulfide bonds. The use of PDI is considered to be extremely efficientspecifically for recombinant proteins having a large number of disulfidebonds. It is also possible to introduce the PDI-encoding gene into atransformed Escherichia coli for the reaction with the recombinantprotein in the Escherichia coli cell.

In accordance with the conventional production method for PDI, it isdifficult to obtain the starting material, but also the amount ofobtainable PDI is limited; therefore, it is desired that a technologyenabling mass production of highly purified PDI will be developed. Inrats and bovines, the PDI genes have already been cloned Nature, 317,267 (1985)!; however, no case of obtaining purified active PDI by theexpression of the PDI gene in Escherichia coli, etc. has been reportedso far. Also, no case of the cloning of PDI of an eukaryote other thanthe above-mentioned eukaryotes has been known up to now.

The present inventors have made studies on the biological actions,proteochemical properties, etc. of the protein (T3 binding protein,hereinafter abbreviated T3BP) which binds specifically with the thyroidhormone T3 (3,3',5-triiodothyronine, hereinafter abbreviated T3). T3BPis known to be present in cell membranes and cytoplasm of mammaliancells, and the T3 receptor is known to be present in cell nuclei. Someof the present inventors succeeded in preparing an antibody againstpurified T3BP as obtained from bovine liver cell membranes Horiuchi, R.and Yamauchi, K.: in Gunma Symposia on Endocrinology, 23 (Center forAcademic Publication Japan, Tokyo: VNU Science Press, BV. Utrecht), pp.149-166 (1986)!, and have endeavored to clone the gene which encodesT3BP protein by means of various genetic engineering techniques, thusfinding that the bovine liver T3BP's cDNA obtained using the aboveanti-T3BP antibody as the probe is encoding bovine PDI proteinInternational Application Number: PCT/JP87/00058; filing date of theinternational application: Jan. 28, 1987!.

In general, the proteins of animals which are more closely related tohumans have extremely high homology in amino acid sequences with thehuman proteins; even most of the portions of different amino acids arederived by one-point mutation of the codons. It is therefore inferredthat the DNA sequence of the above-mentioned bovine T3BP (PDI) geneextremely resembles the DNA sequence of the human PDI gene. The presentinventors then found that human PDI is produced by cloning from humancells the human PDI gene using a part of the bovine PDI gene as the DNAprobe, constructing a recombinant DNA containing said human PDI gene,and cultivating the transformant which has resulted from transformationwith said DNA. The present inventors have made further investigationsbased on these findings to complete this invention.

MEANS OF SOLVING THE PROBLEMS

The present invention provides (1) a recombinant DNA containing the basesequence (I) which encodes the amino acid sequence of FIG. 3, (2) atransformant which has resulted from transformation with a vectorcarrying the base sequence (I), (3) a polypeptide containing the aminoacid sequence of FIG. 3 hereinafter abbreviated human PDI or human PDI(II)!, and (4) a method for producing human PDI characterized in that atransformant which has resulted from transformation with a vectorcarrying the base sequence (I) is cultivated to allow the transformantto produce and accumulate in the culture human PDI, which is thenrecovered.

The base sequence (I) may be any base sequence, as long as it encodesthe amino acid sequence of FIG. 3, but it is preferable that the basesequence be the base sequence from 104 (the 1st base G of the codonwhich encodes the +1 amino acid Ala) to 1573 (the 3rd base G of thecodon which encodes the +490 amino acid Leu) as presented on the lowerline of FIG. 5. Also preferred is the base sequence from 104 to 1573 aspresented on the lower line of FIG. 5 with a translational start codonATG or the base sequence from 50 (the 1st base A of the codon whichencodes the amino acid -18 Met) to 103 (the 3rd base C of the codonwhich encodes the amino acid -1 Asp) added thereto at a site upstreamfrom its 5' end. Preferred examples also include the base sequence from104 to 1573 as presented on the lower line of FIG. 5 with GAC or ATGGACadded thereto at a site upstream from its 5' end, the base sequence from107 to 1573 as presented on the lower line of FIG. 5, and the basesequence from 107 to 1573 as presented on the lower line of FIG. 5 withATG added thereto at a site upstream from its 5' end.

Examples of the human PDI (II) include the polypeptide represented bythe amino acid sequence of FIG. 3, the polypeptide represented by theamino acid sequence of FIG. 3 with Met or the peptide represented by theamino acid sequence from -18 to -1 as presented on the upper line ofFIG. 5 added thereto at its N-terminal, the polypeptide represented bythe amino acid sequence of FIG. 3 with Asp or Met-Asp added thereto atits N-terminal, the polypeptide represented by the amino acid sequenceof FIG. 5 with the amino acid +1 Ala as presented on an upper line ofFIG. 5 eliminated therefrom, and the polypeptide represented by theamino acid sequence of FIG. 5 with the amino acid +1 Ala as presented onan upper line of FIG. 5 replaced by Met. These peptides may besugar-linked glycoproteins, and may also be fused proteins with otherpolypeptides.

An expression vector carrying the base sequence which encodes the humanPDI polypeptide for the present invention can, for example, be producedby (i) separating a mRNA which encodes human PDI, (ii) synthesizing fromsaid RNA a single-stranded complementary DNA (cDNA) and then adouble-stranded DNA, (iii) inserting said complementary DNA in a phageor plasmid, (iv) using the obtained recombinant phage or plasmid totransform the host, (v) cultivating the obtained transformant, and thenisolating the phage or plasmid carrying the desired DNA from thetransformant by an appropriate method such as immunoassay using theanti-T3BP antibody, or plaque hybridization or colony hybridizationusing a radiolabeled probe, (vi) cleaving the desired cloned DNA fromthe phage or plasmid, and (vii) ligating said cloned DNA to thedownstream of a promoter in a vehicle.

The mRNA for encoding human PDI can be obtained from various humanPDI-producing cells, such as human liver cells and placenta cells.

The methods of preparing RNA from human PDI-producing cells include theguanidine thiocyanate method Chirgwin, J. M. et al.: Biochemistry, 18,5294 (1979)!.

Using the RNA thus obtained as the template and reverse transcriptase,the cDNA is synthesized in accordance with, for example, the method ofOkayama, H. et al. Mol. Cell. Biol., 2, 161 (1982) and ibid, 3, 280(1983)! or the method of Gubler, U. and Hoffman, B. J. Gene, 25, 263(1983)!, and the cDNA thus obtained is inserted in the plasmid or phage.

As examples of plasmids to which the cDNA is inserted, there arementioned Escherichia coli-derived plasmids pBR322 Gene, 2, 95 (1977)!,pBR325 Gene, 4, 121 (1978)!, pUC12 Gene, 19, 259 (1982)!, and pUC13Gene, 19, 259 (1982)!, and Bacillus subtilis-derived plasmid pUB110Biochem. Biophys. Res. Commun., 112, 678 (1983)!, but any other plasmidcan also be used, as long as it is replicated and carried by the host.As examples of phage vectors to which the cDNA is inserted, there ismentioned λgt11 Young, R., and Davis, R., Proc. Natl. Acad. Sci. USA,80, 1194 (1983)!, but any other phage vector can be used, as long as itis capable of proliferating in the host.

The methods of inserting the cDNA into the plasmid include the methoddescribed in Maniatis, T. et al., "Molecular Cloning", Cold SpringHarbor Laboratory, p. 239 (1982). The methods of inserting the cDNA intothe phage vector include the method of Hyunh, T. V. et al. DNA Cloning,A Practical Approach, 1, 49 (1985)!. The plasmid or phage vector thusobtained is then introduced into an appropriate host such as Escherichiacoli or Bacillus subtilis.

Examples of such Escherichia coli include Escherichia coli K12 DH1 Proc.Natl. Acad. Sci. USA, 60, 160 (1968)!, JM103 Nucl. Acids Res., 9, 309(1981)!, JA221 J. Mol. Biol., 120, 517 (1978)!, HB101 J. Mol. Biol., 41,459 (1969)!, and C600 Genetics, 39, 440 (1954)!.

Examples of such Bacillus subtilis include Bacillus subtilis MI 114Gene, 24, 255 (1983)! and 207-21 J. Biochem., 95, 87 (1984)!.

The methods of transforming the host with the plasmid include thecalcium chloride method and calcium chloride/rubidium chloride methoddescribed in Maniatis, T. et al., "Molecular Cloning", Cold SpringHarbor Laboratory, p. 249 (1982). Also, the phage vector can, forexample, be introduced into grown Escherichia coli by means of the invitro packaging method.

From the transformants thus obtained, the desired clone is selected bywell-known methods such as the colony hybridization method Gene, 10, 63(1980)!, the plaque hybridization method Science, 196, 180 (1977)!, andthe DNA base sequence determination method Proc. Natl. Acad. Sci. USA,74, 560 (1977); Nucl. Acids Res., 9, 309 (1981)!.

Thus, the microorganism carrying the vector having the cloned DNAcontaining the base sequence which encodes human PDI is obtained.

The plasmid pT3BP-3 carried by Escherichia coli K12 DH5α/pT3BP-3 asobtained in Example 1 below has a DNA containing a base sequence whichencodes human PDI. FIG. 4 shows cleavage sites of said DNA withrestriction enzymes. As shown in FIG. 4, said DNA has a total length ofabout 2.4 Kbp, and is cut into fragments by the restriction enzymes BamHI, EcoR I, Cla I, Hind III, and Pst I.

The plasmid or phage vector is then isolated from the microorganism.

The methods of said isolation include the alkaline extraction methodBirmboim. H. C. et al.: Nucl. Acids Res., 1, 1513 (1979)!.

The above-mentioned plasmid or phage vector carrying the cloned DNAcontaining the base sequence which encodes human PDI may be used as itis, or may be used after digestion with restriction enzymes whennecessary.

The cloned gene may be ligated to a site downstream from a promoter in asuitable vehicle (vector) for expression to thereby give the expressionvector.

The vectors include the above-mentioned Escherichia coli-derivedplasmids (e.g. pBR322, pBR325, pUC12, pUC13, ptrp781), Bacillussubtilis-derived plasmids (e.g. pUB110, pTM5, pC194), yeast-derivedplasmids (e.g. pSH19, pSH15, pGLD906, pGLD906-1, pCDX, pKSV-10),bacteriophages such as λ phage, and animal viruses such as retrovirusesand vaccinia viruses.

Said gene may have ATG as the translational start codon at its 5' end,and may also have TAA, TGA, or TAG as the translational terminationcodon at its 3' end. For expression of said gene, a promoter isconnected thereto at a site upstream from said gene. The promoter to beused in this invention may be any promoter that is appropriate andadapted for the host employed for the expression of said gene.

When the host to be transformed is Escherichia coli, the trp promoter,lac promoter, rec promoter, λP_(L) promoter, lpp promoter, etc. arepreferred. When the host is Bacillus subtilis, the SP01 promoter, SP02promoter, penP promoter, for instance, are preferred. When the host isyeast, the PHO5 promoter, PGK promoter, GAP (GLD) promoter, ADHpromoter, etc. are preferred. In particular, it is preferable that thehost is Escherichia coli and that the promoter is the trp promoter orλP_(L) promoter.

When the host is an animal cell, SV40-derived promoters and retroviruspromoters are usable among others. In particular, SV40-derived promotersare preferable.

The thus-constructed vector carrying the DNA containing the basesequence (I) is used to produce transformants.

Examples of the host include prokaryotes such as Escherichia coli,Bacillus subtilis and actynomycetes, and eukaryotes such as yeasts,fungi and animal cells.

Representative examples of the strains of Escherichia coli and Bacillussubtilis are the same as those mentioned hereinbefore.

Representative examples of the yeasts include Saccharomyces cerevisiaeAH22, AH22R⁻, NA87-11A and DKD-5D.

Representative examples of the animal cells include simian COS-7 andVero cells, Chinese hamster CHO cells and mouse L cells.

The transformation of said Escherichia coli is conducted by the methoddescribed in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) or in Gene, 17,107 (1982), for instance.

The transformation of Bacillus subtilis is conducted by the methoddescribed in Molec. Gen. Genet., 168, 111 (1979), for instance.

The transformation of yeast is conducted by the method described inProc. Natl. Acad. Sci. USA, 75, 1929 (1978), for instance.

The transformation of animal cells is conducted by the method describedin Virology, 52, 456 (1973), for instance.

In this manner, transformants as transformed with vectors carrying thebase sequence (I) are obtained.

In cultivating the transformant obtained by transformation ofEscherichia coli, Bacillus subtilis, an actynomycete, yeast or fungus asthe host, a liquid medium is suitable for the cultivation, and themedium contains substances required for the growth of saidtransformants, for example carbon sources, nitrogen sources andinorganic nutrients. Glucose, dextrin, soluble starch and sucrose, forinstance, may serve as carbon sources. The nitrogen sources may includeorganic or inorganic substances such as ammonium salts, nitrate salts,corn steep liquor, peptone, casein, meat extract, soybean cake andpotato extract. The inorganic nutrients may include calcium chloride,sodium dihydrogen phosphate and magnesium chloride.

The medium preferably has a pH of about 5 to 8.

When the host is Escherichia coli, M9 medium containing glucose andcasamino acids (Miller: J. Exp. Mol. Genet., p. 431, Cold Spring HarborLaboratory, N.Y., 1972), for instance, is a preferable medium for use.The cultivation is normally carried out at about 14° to 43° C. for about3 to 24 hours, and aeration and/or stirring may be conducted whennecessary.

When the host is Bacillus subtilis, the cultivation is normally carriedout at about 30° to 40° C. for about 6 to 24 hours, and aeration and/orstirring may be conducted when necessary.

When a transformant obtained from yeast as the host is cultivated,Burkholder's minimum medium Bostian, K. L. et al.: Proc. Natl. Acad.Sci. USA, 77, 4505 (1980)! and its modified low-Pi medium Biochem.Biophys. Res. Commun., 138, 268 (1986)!, for instance, may be used asthe medium. The pH of the medium is preferably adjusted to about 5 to 8.The cultivation is normally carried out at about 20° to 35° C. for about24 to 72 hours, with aeration and/or stirring when necessary.

As the medium to be used in cultivating a transformant obtained from ananimal cell as the host, there are mentioned, for example, MEM mediumScience, 122, 501 (1952)!, DMEM medium Virology, 8, 396 (1959)!, RPMI1640 medium J. Am. Med. Assoc., 199, 519 (1967)! and 199 medium Proc.Soc. Exp. Biol. Med., 73, 1 (1950)!, which further contain about 5 to20% fetal calf serum. The medium preferably has a pH of about 6 to 8.The cultivation is normally carried out at about 30° to 40° C. for about15 to 60 hours, with aeration and/or stirring when necessary.

The human PDI protein of this invention is intracellularly orextracellularly produced and accumulated. For extracting intracellularPDI from the culture, there can be used as appropriate methods such as amethod comprising collecting the cells by a known method aftercultivation and suspending the cells in a buffer containing a proteindenaturant such as guanidine hydrochloride or urea or a surfactant suchas Triton-100, followed by centrifugation to thereby give a supernatantcontaining the human PDI, or a method comprising disrupting the cells byglass beads, by treatment with ultrasonication, by treatment with anenzyme such as lysozyme or by the freeze-thawing method, followed bycentrifugation to thereby give a supernatant containing the human PDI.

For separating and purifying the human PDI from the supernatant or theextracellularly produced and accumulated human PDI, well-known methodsof separation and purification may be used in appropriate combination.Examples of the known methods of separation and purification includemethods based on differences in solubility such as salting-out andsolvent precipitation; methods based mainly on differences in molecularweight such as dialysis, ultrafiltration, gel filtration andSDS-polyacrylamide gel electrophoresis; methods based on differences incharge such as ion exchange chromatography; methods based on specificaffinity such as affinity chromatography; methods bases on differencesin hydrophobicity such as reverse phase high performance liquidchromatography; methods based on differences in isoelectric point suchas isoelectric electrophoresis; and methods based on specific adsorptionsuch as hydroxyapatite chromatography. Specifically, ion exchangechromatography with diethylaminoethyl (DEAE) cellulose, DEAE Toyopearl,carboxymethyl (CM) cellulose or CM Toyopearl is efficient for thepurification of human PDI protein because said protein is thought of asan acidic protein.

The activity of the human PDI protein obtained as above (PDI activity)can be determined by measuring the rate of conversion of reduced andscrambled ribonucleases as the substrate to active ribonucleasesBiochem. J., 159, 377 (1976); Proc. Natl. Acad. Sci. USA, 83, 7643(1986); Methods in Enzymology, 107, 281 (1984); J. Biol. Chem., 234,1512 (1959)!.

ACTION AND EFFECT

The human PDI protein of the present invention catalyzes the exchangereaction between sulfhydryl and disulfide in any desired protein forformation of the most stable natural type disulfide bonds. Morespecifically, said protein, in the presence of dissolved oxygen, anoxidized glutathione (GSSG)-reduced glutathione (GSH) mixture or areducing agent such as dithiothreitol (DTT), 2-mercaptoethanol orascorbic acid, acts on a reduced protein to catalyze a reaction by whichnatural type disulfide bonds are formed, or acts on an oxidized proteinhaving disulfide bonds, specifically non-natural type disulfide bonds tocatalyze a reaction by which natural type disulfide bonds are re-formed.

Therefore, in producing a protein having disulfide bonds in itsmolecules by means of genetic recombination technology, specificallywhen the host with a recombinant DNA is a cell of a prokaryote such asEscherichia coli, Bacillus subtilis, or Bacillus brevis, human PDIprotein can be used to efficiently form natural type disulfide bonds inthe relevant protein molecule. Such proteins include cytokines such asinterferon-α, interferon-β, interferon-γ, interleukin-1, interleukin-2,B-cell growth factor (BGF), B-cell differentiating factor (BDF),macrophage activating factor (MAF), lymphotoxin (LT), and tumor necrosisfactor (TNF); peptide protein hormones such as transforming growthfactor (TGF), erythropoietin, epithelial cell growth factor (EGF),fibroblast growth factor (FGF), insulin, and human growth hormone;pathogenic microbial antigen proteins such as hepatitis B virusantigens; enzymes such as peptidase and lysozyme; and blood proteincomponents such as human serum albumin and immunoglobulin.

Said PDI protein used for this purpose may be a purified one, or may bea partially purified one; the purpose can be accomplished simply byallowing said PDI protein to act directly on the above-mentionedintracellularly or extracellularly produced and accumulated desiredprotein or a purified standard sample thereof. It is also possible toconduct the reaction using a transformant which has been double infectedwith a recombinant DNA which encodes one of the above-mentioned desiredproteins and the recombinant DNA which encodes said PDI protein.

The symbols used in the specification and drawings have the meanings asmentioned in Table 1.

                  TABLE 1    ______________________________________    PBS          Phosphate buffered saline    DNA          Deoxyribonucleic acid    cDNA         Complementary deoxyribonucleic acid    A            Adenine    T            Thymine    G            Guanine    C            Cytosine    RNA          Ribonucleic acid    mRNA         Messenger RNA    dATP         Deoxyadenosine triphosphate    dTTP         Deoxythymidine triphosphate    dGTP         Deoxyguanosine triphosphate    dCTP         Deoxycytidine triphosphate    ATP          Adenosine triphosphate    EDTA         Ethylenediamine tetraacetic acid    SDS          Sodium dodecyl sulfate    Gly          Glycine    Ala          Alanine    Val          Valine    Leu          Leucine    Ile          Isoleucine    Ser          Serine    Thr          Threonine    Cys          Cysteine    Met          Methionine    Glu          Glutamic acid    Asp          Aspartic acid    Lys          Lysine    Arg          Arginine    His          Histidine    Phe          Phenylalanine    Tyr          Tyrosine    Trp          Tryptophan    Pro          Proline    Asn          Asparagine    Gln          Glutamine    ______________________________________

The amino acid sequence of the PDI protein of this invention may bepartially (up to about 5%) modified (including addition, elimination,and substitution by other amino acids).

EXAMPLES

The present invention will now be described in more detail by means ofthe following reference examples and working examples, which are not tobe construed as limitations on the invention.

Reference Example 1 (Preparation of Bovine Liver mRNA-derived cDNALibrary)

The RNAs were extracted from a bovine liver by the guanidineisothiocyanate method Chirgwin, J. M. et al.: Biochemistry, 18, 5294(1979)!. From these RNAs, poly (A) RNA was purified by oligo dTcellulose column chromatography Aviv and Leder: Proc. Natl. Acad. Sci.USA, 69, 1408 (1972)!.

Using this poly (A) RNA as the template, the cDNA was prepared by themethod of Gubler, U. and Hoffman, B. J. Gene, 25, 263 (1983)!; then, anEcoR I liner was added to the above cDNA by the method of Huynh, T. V.et al. DNA cloning I, a practical approach, 1, 49 (1985)!, and the cDNAwas cloned to the EcoR I site in λgt11 to thereby prepare a cDNAlibrary.

Reference Example 2 (Isolation of Plasmid Containing Bovine PDI cDNA andDetermination of its Base Sequence)

Escherichia coli Y1096, after being infected with the cDNA library ofλgt11 obtained in Reference Example 1, was spread on an L-broth softagar plate. Upon the appearance of plaques, a nitrocellulose filtercontaining IPTG (isopropylthiogalactoside) was placed on the plate, andincubation was conducted for 3 hours. The nitrocellulose filter was thenseparated and washed with TBS buffer (50 mM Tris, pH 7.9, 150 mM NaCl),after which it was dipped in a 3% gelatin solution.

The nitrocellulose filter thus treated was immersed in a solution ofantibodies against T3 binding protein (anti-BLT₃ R) Horiuchi, R. andYamauchi, K.: Gunma Symposia on Endocrinology, 23, P. 149 (1986)! forantigen-antibody reaction. Said filter was then washed with distilledwater and TBS buffer, and then reacted with the secondary antibody toselect the coloring positive clone λgt11 T3BP-1. The cDNA in the λgt11T3BP-1 was cleaved with EcoR I and re-cloned to the EcoR I site in theplasmid pUC19 Gene, 33, 103 (1985)! to thereby prepare the plasmidpT3BP-1. Using the fragment cleaved with the restriction enzymes EcoR Iand Pvu II from the cDNA region contained in said plasmid as the DNAprobe, 5×10⁵ clones of the cDNA library described in Example 1 werere-screened to thereby select the positive clones.

From one of the clones thus obtained, namely λgt11 T3BP-2, the cDNA wascleaved with Sac I-Kpn I and re-cloned to the Sac I-Kpn I site in theplasmid pUC19 to thereby prepare the plasmid pT3BP-2. (The EcoR Irecognition site in the 5' terminal side of the cDNA cloned to λgt11T3BP-2 had been destroyed.)

E. coli K12 DH5α was transformed with the plasmid pT3BP-2; from theresulting transformant E. coli K12 DH5α/pT3BP-2, the plasmid pT3BP-2 wasextracted and purified by the alkali method Birnboim, H. C. and Doly,J.: Nucl. Acids Res., 11, 1513 (1979)!. The cDNA region contained inthis plasmid has a total length of about 2.8 Kbp. FIG. 1 shows aschematized cleavage map of the cDNA region with restriction enzymes.

The base sequence of this cDNA region was determined by thedideoxy-nucleotide chain termination method Messing, J. et al.: Nucl.Acids Res., 9, 309 (1981)!. FIG. 2 shows the amino acid sequence deducedfrom the determined base sequence.

The amino acid sequence of said polypeptide has homology of more than90% with that of rat PDI, but no homology with the amino acid sequenceof thyroxine binding globulin (TBG), thyroxine binding prealbumin (TBPA)or C-erbA protein, all of which are capable of binding with thyroidhormone.

Escherichia coli K12 DH5α/pT3BP-2, carrying the plasmid pT3BP-2, hasbeen deposited at the Institute for Fermentation, Osaka (IFO) under theaccession number IFO 14563, and has also been deposited at theFermentation Research Institute (FRI), Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, Japan underthe accession number FERM BP-1595.

The EcoR I-Pvu II fragment of the cDNA carried by pT3BP-1 can also beobtained by treatment of pT3BP-2 with Kpn I and Sac I followed bytreatment with EcoR I and Pvu II.

Example 1 (Isolation of Plasmid Containing Human PDI cDNA andDetermination of its Base Sequence)

A λgt11 cDNA library produced on the basis of human placental mRNA(purchased from Toyobo Co., Ltd., Japan), using Escherichia coli Y1096as the host, was spread on 4 soft agar plates in an amount of about1×10⁵ clones per plate, and these were transferred to nitrocellulosefilters (Millipore's HATF filters). The phages on these filters werethen lysed with a 0.5N NaOH solution, and the exposed and denaturedphage DNAs were immobilized on the filters while drying (Maniatis, etal.: "Molecular Cloning", Cold Spring Harbor Laboratory, P. 320, 1982).Separately, the DNA fragment obtained by cleaving with the restrictionenzymes EcoR I and Pvu II the cDNA portion contained in the plasmidpT3BP-1 described in Reference Example 2 was labeled with ³² P by thenick translation method (Maniatis et al.: ibid, P. 109, ) for use as theprobe.

Hybridization reaction was carried out between the labeled probe and theDNA-immobilized filters in 10 ml of a mixture containing the labeledprobe, 5×SSPE 0.9M NaCl, 50 mM sodium phosphate buffer (pH 7.4), 5 mMEDTA!, 50% formamide, 5×Denhardt's solution, 0.1% SDS and 100 μg/mldenatured salmon sperm DNA at 42° C. for 16 hours. After the reaction,the filters were washed twice with 2×SSC (1×SSC=0.15M NaCl, 0.015Msodium citrate)-0.1% SDS solution at room temperature for 30 minutes,and further washed twice with 1×SSC-0.1% SDS solution at 68° C. for 30minutes. After the washed filters were dried, radioautograms were taken,and clones reacting with the probe were picked up. The phage DNA wasextracted from the clone thus obtained, namely λgt11 T3BP-3, by themethod of Davis et al. (Davis et al.: "Advanced Bacterial Genetics",Cold Spring Harbor Laboratory, 1980). The cDNA was then cut out fromλgt11 T3BP-3 with EcoR I, and re-cloned to the EcoR I site in theplasmid pUC19 to thereby construct the plasmid pT3BP-3. E. coli K12 DH5αwas transformed with the plasmid pT3BP-3; from the obtainedtransformant, namely E. coli K12 DH5α/pT3BP-3, the plasmid pT3BP-3 wasextracted and purified by the alkaline extraction method Birnboim, H. C.and Doly, J.: Nucl. Acids Res., 11, 1513 (1979)!. The cDNA portioncontained in this plasmid has a total length of about 2.4 Kbp. FIG. 4shows a schematized cleavage map with restriction enzymes of the cDNAportion.

The base sequence of this cDNA portion was determined by thedideoxynucleotide chain termination method Messing, J. et al.: Nucl.Acids Res., 9, 309 (1981)!. FIG. 5 shows the amino acid sequence deducedfrom the determined base sequence.

The amino acid sequence of said polypeptide has homology of more than90% with that of rat PDI, but no homology with the amino acid sequenceof thyroxine binding globulin (TBG), thyroxine binding prealbumin (TBPA)or C-erbA protein, all of which are capable of binding with thyroidhormone.

Escherichia coli K12 DH5α carring the plasmid pT3BP-3 (Escherichia coliK12 DH5α/pT3BP-3) has been deposited at the IFO under the accessionnumber IFO 14610, and has also been deposited at the FRI under theaccession number FERM BP-1841 (transferred from FERM P-9386).

Example 2 (Construction of Expression Plasmid for Animal Cells)

The DNA of the plasmid pT3BP-3 was cleaved with the restriction enzymeEcoR I, to separate the 2.4 Kbp cDNA as shown in FIG. 4. Separately, theplasmid pTB389, which was obtained by converting the Pst I cleavage sitein the 5' end side and BamH I cleavage site in the 3' end side in theinterleukin-2 gene region of the plasmid pTB106 which was described inJapanese Unexamined Patent Publication No. 63282/1986 into EcoR I sitesand eliminating the interleukin-2 gene region, was cleaved with therestriction enzyme EcoR I and the phosphate group in the 5' end thereofwas eliminated by alkaline phosphatase treatment. The resulting fragmentand the above cDNA were mixed and ligated together by T4 DNA ligase tothereby construct the plasmid pTB745 (FIG. 6).

Example 3 (Expression of Human PDI-encoding Gene in Animal Cells)

Simian COS-7 cells were cultured in the manner of monolayer culture onDMEM medium containing 10% fetal calf serum, and the medium was thenreplaced with a fresh one of the same medium. Four hours after theexchange of medium, calcium phosphate gel containing 10 μg of the DNA ofthe plasmid pTB745 was prepared in accordance with a known method Grahamet al.: Virology, 52, 456 (1973)! and added to the cells to thereby givepTB745-infected COS-7 cells. Further 4 hours later, the cells weretreated with glycerol, and then cultivation of the pTB745-infected COS-7cells was continued on a medium containing 0.5% fetal calf serum.

After 48 hours, the pTB745-infected COS-7 cells were fixed with PBScontaining 3.5% formalin at room temperature for 15 minutes. The cellswere treated with PBS containing 0.1% saponine at room temperature for10 minutes, and then reacted with the above-mentioned anti-bovine T3BPrabbit antibody at 4° C. for 2 hours. After thoroughly washing out theunreacted antibodies with PBS, FITC-labeled anti-rabbit IgG sheepantibodies were reacted with the cells overnight, and the cells wereobserved by means of a fluorescence microscope. The results are shown inFIG. 7. Evidently stronger fluorescence was observed in the cells intowhich the human PDI gene were introduced than in the control COS cells;it was found that human PDI protein was synthesized in the COS cells.

Example 4 (Construction of Plasmid for Expression of Human PDI Gene inEscherichia coli)

The plasmid pT3BP-3 obtained in Example 1 as mentioned above andcontaining the human PDI cDNA was cleaved with the restriction enzymeAva I, to obtain a 0.85 Kbp DNA fragment containing the first half ofthe human PDI-encoding region. This DNA fragment was reacted with DNApolymerase (Klenow fragment) in the presence of dATP, dCTP, dGTP anddTTP to thereby render the Ava I cleavage sites blunt. This DNA fragmentwas ligated with phosphorylated synthetic oligonucleotides, ^(5')AATTCTATGGCGC^(3') and ^(5') GCGCCATAG^(3'), in the presence of T4 DNAligase, and cleaved with EcoRI and ClaI to prepare a 0.49 Kbp DNAfragment. Separately, the plasmid pT3BP3 was cleaved with Cla I and PstI to prepare a 1.47 Kbp DNA fragment containing the second half of thePDI-encoding region. The DNA of the trp promoter-containing plasmidptrp781 Kurokawa, T. et al.: Nucleic Acids Res., 11, 3077-3085 (1983)!was cleaved with EcoR I and Pst I, to isolate and an about 3.2 Kbp DNAfragment containing the trp promoter, the tetracycline resistance geneand the plasmid replication origin. This 3.2 Kbp EcoR I-Pst I DNAfragment was ligated with the above-mentioned 0.49 Kbp EcoR I-Cla I DNAfragment containing the human PDI-encoding gene and the 1.47 Kbp Cla I-Pst I DNA fragment by T4 DNA ligase, to construct a human PDIexpression plasmid, pTB766 (FIG. 8). This plasmid was used to transformEscherichia coli K12 DH1 and MM294 strains to give transformantscarrying the plasmid pTB766, namely Escherichia coli K12 DH1/pTB766 andMM294/pTB766.

Escherlchia coli K12 MM294 carrying the plasmid pTB766 (Escherichia coliMM294/pTB766) has been deposited at the IFO under the accession numberIFO 14611, and has also been deposited at the FRI under the accessionnumber FERM BP-1842 (transferred from FERM P-9391). Escherichia coli K12DH1 carrying the plasmid ptrp781 (Escherichia coli DH1/ptrp781) has beendeposited at the IFO under the accession number IFO 14546 and at the FRIunder the accession number FERM BP-1591 (transferred from FERM P-9055).

Example 5 (³⁵ S-methionine Labeling and Immunoprecipitation Reaction ofEscherichia coli)

Escherichia coli MM294 or DH1 carrying the expression plasmid pTB766 wascultivated in M9 medium containing 8 μg/ml tetracycline, 0.2% casaminoacids and 1% glucose at 37° C. When the Klett value was 200, IAA(3β-indolylacrylic acid) was added to a concentration of 25 μg/ml. Twohours later, ³⁵ S-methionine was added to an activity of 15 μCi/ml, andthe synthesized protein was labeled during the following 30 minutes.After the labeling, the cells were harvested and washed with a 0.15MNaCl solution 10% sucrose solution in 10 mM Tris-HCl, pH 8.0, ofone-fifth volume to the culture medium. To this suspension were addedphenylmethylsulfonyl fluoride (PMSF) to 1 mM, NaCl to 0.2M and EDTA to10 mM, and lysozyme was further added to 150 μg/ml. After the reactionwas carried out at 0° C. for 1 hour , the suspension was treated at 31°C. for 2 minutes, and then ultrasonicated for a while (about 10seconds). The sonication product was centrifuged to give a cell extract.To this cell extract was added the anti-bovine T3BP rabbit antibodyHoriuchi, R. and Yamauchi, K.: Gunma Symposia on Endocrinology, 23, 149(1986)!, and the reaction was carried out at 4° C. overnight.Staphylococcal cells (Protein A) were then further added, and the wholemixture was allowed to stand at 0° C. for 3 hours to thereby bind theantigen-antibody conjugate with the cells. The cells were washedrepeately by centrifugation with a solution of 5 mM EDTA, 150 mM NaCl,0.05% Nonidet P-40 (Shell Oil) and 1 mg/ml ovalbumin in 50 mM Tris-HCl,pH 7.4, and then treated at 100° C. for 5 minutes in an electrophoresissample preparation solution (2% SDS, 5% 2-mercaptoethanol, 0.001%bromophenol blue and 10% glycerol in 0.0625M Tris-HCl, pH 6.8) tothereby elute the conjugate. The obtained immunoprecipitate was analyzedby means of 10 to 20% gradient SDS-polyacrylamide gel electrophoresis.After the electrophoresis, the gel was fixed with a 50% trichloroaceticacid solution, and a radioautogram was taken by the fluorographytechnique. From the results shown in FIG. 9, it is evident that PDI ofthe expected size (about 60 kDa) was produced in either of the 2 strainscarrying the expression plasmid, namely Escherichia coli DH1/pTB766 andMM294/pTB766.

Example 6 (Construction of Plasmid for Expression of Human PDI Gene inYeasts)

The plasmid pT3BP-3 obtained in Example 1 as mentioned above andcontaining the human PDI cDNA was digested with the restriction enzymeSac I and rendered blunt-ended by T4 DNA polymerase reaction. The Sal Ilinker GGTCGACC was joined to the above cleavage product. The ligationproduct was further digested with the restriction enzymes Sal I and ClaI, to prepare a 0.65 Kbp DNA fragment. Also, the DNA of the plasmidpTB745 described in Example 2 was digested with the restriction enzymesSca I and Cla I, to isolate a 3.8 Kbp DNA fragment. Further, the DNA ofthe plasmid pGLD906-1 Biochem. Biophys. Res. Commun., 138, 268 (1986)!was digested with the restriction enzymes Sal I and Sca I, to prepare a6.2 Kbp DNA fragment. This DNA fragment was ligated with theabove-mentioned 2 DNA fragments by means of T4 DNA ligase, to constructa plasmid for expression in yeasts, pTB767 (FIG. 10). This plasmid wasused to transform AH22R-strain to give a yeast strain carrying theplasmid pTB767, namely Saccharomyces cerevisiae AH22R⁻ /pTB767.

Saccharomyces cerevisiae AH22R⁻ carrying the plasmid pTB767(Saccharomyces cerevisiae AH22R⁻ /pTB767) has been deposited at the IFOunder the accession number IFO 10425, and has also been deposited at theFRI under the accession number FERM BP-1843 (transferred from FERMP-9603).

Example 7 (³⁵ S-methionine Labeling and Immunoprecipitation Reaction ofYeast)

The yeast strain AH22R⁻ carrying the expression plasmid pTB767 wascultivated in low-Pi medium Biochem. Biophys. Res. Commun., 138, 268(1986)! at 37° C. overnight, and then ³⁵ S-methionine was added to 20μCi/ml to label the synthesized protein. After the labeling, the cellswere harvested and washed with a 0.15M NaCl solution, and 7M guanidinehydrochloride of one-fifth volume to the culture medium was added. Afterthe cells were lysed at 0° C. for 1 hour, the lysate was centrifuged at10,000 rpm for 10 minutes. The resulting supernatant was dialyzedagainst a solution containing 10 mM Tris.HCl (pH 8.0), 1 mM EDTA, 200 mMNaCl and 1 mM PMSF to give a cell extract. To this cell extract wasadded the anti-bovine T3BP rabbit antibody (described in Example 5), andthen the procedure described in Example 5 was taken to give animmunoprecipitate, which was then analyzed by SDS-polyacrylamide gelelectrophoresis and radioautography. As a result, there was synthesizeda polypeptide specific to the yeast strain AH22R⁻ /pTB767 carrying theexpression plasmid; it was demonstrated that human PDI was produced inthe yeast.

The composition of the used low-Pi medium was as follows: Low-Pi mediumper 1 l!

    ______________________________________    KC1                     1.5    g    Glucose                 20     g    Asparagine              2      g    L-histidine             100    mg    KI(1 mg/ml)             100    ul    MgSO.sub.4.7H.sub.2 O (500 mg/10 ml)                            10     ml    CaCl.sub.2.2H.sub.2 O) (330 mg/10 ml)                            10     ml    Trace element solution (a)                            1      ml    Vitamine solution (b)   1      ml    Trace element solution (a)  per 1 l!    CuSO.sub.4.5H.sub.2 O)  40     mg    FeSO.sub.4.7H.sub.2 O)  250    mg    MnSO.sub.4.4H.sub.2 O)  40     mg    (NH.sub.4).sub.3 PO.sub.4.12MoO.sub.3.3H.sub.2 O)                            20     mg    ZnSO.sub.4.7H.sub.2 O   310    mg    Vitamine solution (b)  per 1 l!    Inositol                10     g    Thiamine                200    mg    Pyridoxine              200    mg    Calcium pantothenate    200    mg    Niacin                  200    mg    Biotin                  2      mg    ______________________________________

Example 8 (Purification of PDI Derived From Escherichia coliMM294/pTB766)

Escherichia coli MM294 carrying the expression plasmid pTB766 obtainedin Example 4 was cultivated in 1-liter of a medium containing 5 mg/ltetracycline, 10 g/l Bacto-trypton, 5 g/l Bacto-yeast extrafct and 5 g/lNaCl at 37° C. for 11 hours. The culture was then transferred to20-liter of M-9 medium containing 2 mg/l vitamine B₁, 10 g/l glucose and10 g/l casamino acids, and the culture was maintained with agitation andaeration at 37° C. for further 9 hours. After cultivation, the cells(385 g by wet weight) were collected by centrifugation and stored at-80° C. until used.

The cells (30 g by wet weight) were suspended in 150 ml of 20 mMTris-HCl buffer (pH 7.4) containing 0.15M NaCl, 10 mMethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonylfluoride (PMSF), and 0.1 mM (p-amidinophenyl)methanesulfonylfluoride-HC1 (APMSF). The suspension was subjected to successivesonication (1 min×4 times) at 0° C. and centrifuged to give 156 ml of acrude extract.

The crude extract was applied to a Cellulofine GCL-2000-sf (SeikagakuKogyo Co., Ltd., Tokyo, Japan) column (6×102 cm, 2880 ml) which had beenpreviously equilibrated with 10 mM sodium phosphate buffer (pH 6.8)containing 0.15N NaCl, 1 mM CaCl₂ and 0.1 mM APMSF. The column wasdeveloped with the same buffer and the eluates (325 ml) containing PDIwere collected.

The eluates thus obtained were applied to a hydroxyapatite (Bio-Rad,California, U.S.A.) column (1.5×14 cm, 25 ml) which had been previouslyequilibrated with 10 mM sodium phosphate buffer (pH 6.8) containing0.145M NaCl, 1 mM CaCl₂ and 0.1 mM APMSF. The column was washed with thesame buffer and then developed with a linear gradient of sodiumphosphate concentration. The gradient was produced by adding 200 ml of300 mM sodium phosphate buffer (pH 6.8) to 200 ml of 10 mM sodiumphosphate buffer (pH 6.8). Eluates containing PDI were collected.

The eluates were dialyzed overnight against 10 mM sodium phosphatebuffer (pH 6.8) containing 0.1 mM APMSF. The dialyzed solution was thenapplied to a DEAE-Toyopearl (Tosoh, Tokyo, Japan) column (1.5×15 cm,26.5 ml) equilibrated with the same buffer. The column was washed withthe same buffer and then developed with a linear gradient of NaClconcentration. The gradient was produced by adding 200 ml of 10 mMsodium phosphate buffer (pH 6.8) containing 0.5M NaCl and 0.1 mM APMSFto 200 ml of 10 mM sodium phosphate buffer (pH 6.8) containing 0.1 mMAPMSF. Fractions containing PDI were collected.

The fractions containing PDI were dialyzed against 10 mM sodiumphosphate buffer (pH 6.8) containing 0.1 mM APMSF. The dialyzed solutionwas applied to a DEAE-NPR HPLC column (Tosoh, Tokyo, Japan; 0.46×3.5 cm)which had been previously equilibrated with 10 mM sodium phosphatebuffer (pH 6.8) containing 0.09M NaCl. Proteins were eluted byincreasing the NaCl concentration from 0.09M to 0.39M in a period of 30minutes. Fractions containing PDI were collected and sterilized byfiltration. Protein concentration, 106 μg/ml; total volume, 17.1 ml;total protein, 1.8 mg.

The final preparation thus obtained was homogeneous as judged bySDS-polyacrylamide gel electrophoresis (FIG. 11).

Example 9 (Alternative Purification Method for PDI Derived FromEscherichia coli MM294/pTB766)

E. coli cells (30 g) obtained in Example 8 were suspended in 160 ml of20 mM Tris-HCl buffer (pH 7.4) containing 0.15M NaCl, 10 mM EDTA, 1 mMPMSF and 0.1 mM APMSF. The cells were disrupted at 0° C. for 30 minutesby glass beads with a diameter of 0.25 to 0.5 mm using a Bead-Beater(Biospec Products Inc., U.S.A.). The suspension was centrifuged and thesupernatant fluid (84 ml) was collected.

The supernatant fluid was successively subjected to the samepurification procedures described in Example 8 to give a homogeneous PDIpreparation. Protein concentration, 111 μg/ml; total volume, 9.0 ml;total protein, 1.0 mg.

Example 10 (Protein-chemical Characterisations of PDI)

The PDI preparation obtained in Example 8 was subjected to the followingprotein-chemical analyses.

(1) Molecular weight:

PDI was subjected to SDS-polyacrylamide gel electrophoresis underreducing conditions Nature 227, 680 (1970)! and the proteins werestained with Coomassie Brilliant Blue R-250. PDI showed a single bandwith an approximate molecular weight of 55,000 (FIG. 11).

(2) Amino acid composition:

The amino acid composition was determined on 24, 48 and 72-hourhydrolysates with 6N-HCl at 110° C. in the presence of 4% thioglycolicacid. Amino acid analysis was performed by the ninhydrin method in aHitachi amino acid analyzer model 835. The values of Ser and Thr wereobtained by extrapolating to zero time hydrolysis, and H-Cys (halfcystine) was determined as cysteic acid on a 24-hour hydrolysate afterperformic acid oxidation. The amino acid composition agreed well withthat deduced from the cDNA base sequence as shown in Table 2.

                  TABLE 2    ______________________________________                 Number of                 residues per                           Values predicted    Amino acid   molecule  from cDNA sequence    ______________________________________    Asp/Asn      54.4      57    Thr          21.8      23    Ser          21.2      23    Glu/Gln      70.7      69    Pro          20.5      21    Gly          29.5      29    Ala          46.6      44    H-Cys        5.5       6    Val          31.1      29    Met          4.5       5    Ile          21.5      23    Leu          41.5      41    Tyr          11.9      12    Phe          34.0      33    Lys          46.6      47    His          11.7      11    Arg          13.6      13    Trp          4.5       5    ______________________________________

(3) Amino terminal amino acid sequence:

The sequence analysis of PDI (102 μg; 1.85 nmole) was performed in agas-phase protein sequencer (model 470A, Applied Biosystems, California,U.S.A.). Phenylthiohydantoin (PTH)-amino acid was determined by HPLC ona Micropak SP-ODS column (varian, U.S.A.). The sequence was identicalwith that deduced from cDNA up to 20 cycles as shown in Table 3. Residuenumbers 8 and 13 were not identified.

                  TABLE 3    ______________________________________            PTH-amino acid detected                             Amino acid predicted    Cycle   Residue   pmole      from cDNA sequence    ______________________________________    1       Met       373        (Met)    2       Ala       720        Ala    3       Pro       277        Pro    4       Glu       319        Glu    5       Glu       379        Glu    6       Glu       353        Glu    7       Asp       303        Asp    8       --        --         His    9       Val       271        Val    10      Leu       581        Leu    11      Val       333        Val    12      Leu       638        Leu    13      --        --         Arg    14      Lys       227        Lys    15      Ser        92        Ser    16      Asn       257        Asn    17      Phe       330        Phe    18      Ala       615        Ala    19      Glu       334        Glu    20      Ala       669        Ala    ______________________________________

(4) Carboxyl terminal amino acid:

The carboxyl terminal amino acid of PDI (151 μg; 2.75 nmoles) washydrazinolysis J. Biochem., 59, 170 (1966)! and the amino acid sdetermined by amino acid analysis. The carboxyl terminal thus determinedwas leucine. The recovery was 46%.

(5) Ultraviolet (UV) absorption spectrum:

PDI showed an UV absorption spectrum with a peak absorption at 280 nm in10 mM sodium phosphate buffer (pH 6.8) (FIG. 12).

Example 11 (Refolding of Scrambled Ribonuclease (RNase) by PDI)

The enzymatic activity of PDI obtained in Example 8 was determined byits ability to refold scrambled RNase in the presence of dithiothreitol(DTT).

(1) PDI:

The homogeneous preparation obtained in Example 8 was used.

(2) Scrambled RNase:

Scrambled RNase was prepared according to the method of Hillson et al.Methods in Enzymology 107, 281-284 (1984)! starting from bovinepancreatic RNase.

(3) Determination of RNase activity:

The activity of RNase was determined by the method of Kalnisky et al. J.Biol. Chem., 234, 1512-1516 (1959)! with yeast poly RNA as substrate.

(4) Buffers:

Buffer A: 100 mM sodium phosphate buffer (pH 7.8) containing 10 mM EDTA;buffer B: 0.2M sodium acetate buffer (pH 5.0); buffer C: 0.1M sodiumacetate buffer (pH 5.0).

(5) Refolding of scrambled RNase:

A test tube containing 10 μl of PDI (111 μg/ml), 168 μl of buffer A and2 μl of 1 mM DTT was placed in a water bath of 30° C. and preincubatedfor 5 minutes. Twenty (20) μl of scrambled RNase (0.5 mg/ml) was addedto the test tube and the whole reaction mixture was kept at 30° C. for60 minutes. The reaction was terminated by adding 800 μl of buffer B.

Refolded and active RNase formed in the reaction mixture was determinedas follows: Two hundred (200) μl of the above reaction mixture wastransferred to a centrifuge tube and preincubated at 37° C. for 5minutes. Two hundred (200) μl of 1% yeast poly RNA which had beenpreincubated at 37° C. for 5 minutes was added to the reaction mixturein the centrifuge tube and the whole mixture was kept at 37° C. for 4minutes. Two hundred (200) μl of 25% perchloric acid solution containing0.75% uranium acetate was added to the reaction mixture and the wholemixture was incubated in a ice-water bath for 5 minutes and centrifugedat 16,000 rpm for 5 minutes. The supernatant fluid was dilutedthirty-fold with distilled water and the absorbance at 260 nm wasdetermined.

PDI caused the refolding of scrambled RNase in the presence of 1×10⁻⁵ MDTT: About 25% of the scrambled RNase was converted into an active andrefolded form under the experimental conditions employed (Table 4).

                  TABLE 4    ______________________________________    Addition      RNase activity (%)    ______________________________________    None          0.9    PDI           0.9    DTT           4.4    PDI + DTT     24.9    ______________________________________

Example 12 (Refolding of Scrambled Recombinant Interleukin-2 (rIL-2) byPDI)

PDI obtained in Example 8 was added to a scrambled rIL-2 solution andthe refolding of scrambled rIL-2 was determined by the increase of IL-2activity.

(1) Scrambled rIL-2:

Scrambled rIL-2 was prepared according to the method of Brawning et al.Anal. Biochem., 155, 123-128 (1986)! starting from homogeneous rIL-2Kato et al., Biochem. Biophys. Res. Commun., 130, 692-699 (1985)!.Scrambled rIL-2 is known to possess little biological activity Brawninget al., Anal. Biochem., 155, 123-128 (1986)!.

(2) Determination of IL-2 activity.

Biological activity of IL-2 was determined by the modified MTT methodusing mouse NKC3 cells reported by Tada et al. J. Immunol. Methods, 93,157-165 (1986)!.

(3) Refolding of scrambled rIL-2:

One hundred and fifty (150) μl of scrambled rIL-2 (200 μg/ml) was addedto a test tube containing varying amount of PDI (final concentration of0, 0.74, 1.85 and 3.70 μg/ml), 1000 μl of 30 mM Tris-acetate buffer (pH9.0), 30 μl of 1 mM DTT and distilled water in a final volume of 2850μl. The whole reaction mixture was incubated at 30° C. for 18 hours.IL-2 activity was determined for each tube by the modified MTT method.

Native rIL-2 was increased with the increase of PDI concentration addedto the reaction mixture (Table 5).

                  TABLE 5    ______________________________________           PDI added                  Active IL-2           (μg/ml)                  (μg/ml)    ______________________________________           0      1.28           0.74   2.37           1.85   2.77           3.70   3.02    ______________________________________

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence of FIG.
 3. 2. The polypeptide according to claim 1, which isproduced by a recombinant microorganism.
 3. The polypeptide according toclaim 2, wherein the microorganism is Escherichia coli.
 4. Thepolypeptide according to claim 1, which comprises the amino acidsequence: ##STR1##
 5. A method for producing a protein of a eukaryoticorganism having a natural type disulfide bond in its molecule, whichcomprises subjecting a protein of a eukaryotic organism produced from arecombinant prokaryotic cell, to a reaction with a polypeptidecomprising the amino acid sequence of FIG.
 3. 6. The method according toclaim 5, wherein the reaction is carried out in the presence of areducing agent.